Shows the layout of samples, controls (e.g., negative controls (NC), inter-plate controls (IPC)), and special wells if any (e.g., sample controls (SC), bridge samples) on the 96 well assay plate.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | SMI A1 Donor01 | SMI A2 Donor02 | SMI A3 Donor03 | SMI A4 Donor04 | SMI A5 Donor05 | SMI A6 Donor06 | SMI A7 Donor07 | SMI A8 Donor08 | SMI A9 Donor09 | SMI A10 Donor10 | SMI A11 PP01 rep01 | SMI A12 IPC rep01 |
| B | SMI B1 Donor11 | SMI B2 Donor12 | SMI B3 Donor13 | SMI B4 Donor14 | SMI B5 Donor15 | SMI B6 Donor16 | SMI B7 Donor17 | SMI B8 Donor18 | SMI B9 Donor19 | SMI B10 Donor20 | SMI B11 PP01 rep02 | SMI B12 IPC rep02 |
| C | SMI C1 Donor21 | SMI C2 Donor22 | SMI C3 Donor23 | SMI C4 Donor24 | SMI C5 Donor25 | SMI C6 Donor26 | SMI C7 Donor27 | SMI C8 Donor28 | SMI C9 Donor29 | SMI C10 Donor30 | SMI C11 PP01 rep03 | SMI C12 IPC rep03 |
| D | SMI D1 Donor31 | SMI D2 Donor32 | SMI D3 Donor33 | SMI D4 Donor34 | SMI D5 Donor35 | SMI D6 Donor36 | SMI D7 Donor37 | SMI D8 Donor38 | SMI D9 Donor39 | SMI D10 Donor40 | SMI D11 PP01 rep04 | SMI D12 IPC rep04 |
| E | SMI E1 Donor41 | SMI E2 Donor42 | SMI E3 Donor43 | SMI E4 Donor44 | SMI E5 Donor45 | SMI E6 Donor46 | SMI E7 Donor47 | SMI E8 Donor48 | SMI E9 Donor49 | SMI E10 Donor50 | SMI E11 PP02 rep01 | SMI E12 NTC rep01 |
| F | SMI F1 Donor51 | SMI F2 Donor52 | SMI F3 Donor53 | SMI F4 Donor54 | SMI F5 Donor55 | SMI F6 Donor56 | SMI F7 Donor57 | SMI F8 Donor58 | SMI F9 Donor59 | SMI F10 Donor60 | SMI F11 PP02 rep02 | SMI F12 NTC rep02 |
| G | SMI G1 Donor61 | SMI G2 Donor62 | SMI G3 Donor63 | SMI G4 Donor64 | SMI G5 Donor65 | SMI G6 Donor66 | SMI G7 Donor67 | SMI G8 Donor68 | SMI G9 Donor69 | SMI G10 Donor70 | SMI G11 PP02 rep03 | SMI G12 NTC rep03 |
| H | SMI H1 Donor71 | SMI H2 Donor72 | SMI H3 Donor73 | SMI H4 Donor74 | SMI H5 Donor75 | SMI H6 Donor76 | SMI H7 Donor77 | SMI H8 Donor78 | SMI H9 Donor79 | SMI H10 Donor80 | SMI H11 PP02 rep04 | SMI H12 NTC rep04 |
Summary of the number and classification of reads for the assay run
| Total reads | Parseable | Parseable Match | Parseable Non-match | Unparseable | Total samples | Total targets | Total samples * targets | Zero values | |
|---|---|---|---|---|---|---|---|---|---|
| Plate 1 | 557,800,321 | 443,499,234 (79.5%) | 320,863,871 (57.5%) | 122,635,363 (22.0%) | 114,301,087 (20.5%) | 96 | 212 | 20,352 | 651 (3.2%) |
Each sample of the assay is spiked with the same concentration of an internal control (IC). This is used both as part of a well normalization procedure and as a method for assessing the uniformity of the assay run.
| Zeros n (%) | Total reads (%) | Mean | SD | CV% | |
|---|---|---|---|---|---|
| Plate1 | 0 (0.0%) | 575,548 (0.2%) | 5,995.3 | 3,314.8 | 55.3% |
IPC samples are pooled plasma controls used both to normalize samples between experiments and to assess performance (e.g., precision) of the assay.
Note: %CV in this table does not exclude values below LOD. See Intra-plate normalization or Inter-plate normalization sections for %CV (before and after normalization) that excludes values below LOD.
| Total count (%) | Mean count per target | Zeros n (%) | CV% mean | CV% median | |
|---|---|---|---|---|---|
| Total | 4,216,763 (1.3%) | 4,972.6 | 20 (2.4%) | 12.9% | 11.8% |
| SMI_A12_IPC_rep01 | 954,338 (0.3%) | 4,501.6 | 5 (2.4%) | ||
| SMI_B12_IPC_rep02 | 983,653 (0.3%) | 4,639.9 | 5 (2.4%) | ||
| SMI_C12_IPC_rep03 | 1,079,936 (0.3%) | 5,094.0 | 5 (2.4%) | ||
| SMI_D12_IPC_rep04 | 1,198,836 (0.4%) | 5,654.9 | 5 (2.4%) | ||
| Median | 1,035,337 (0.3%) | 4,883.7 | 5 (2.4%) |
NC wells are reactions where no sample input is provided (i.e., buffer only). These are used to assess Limit of Detection (LOD) for each target assay and to assess run quality (e.g., background levels).
| Total count (%) | Mean count per target | Max target count | Missing n (%) | |
|---|---|---|---|---|
| NC total | 252,207.00 (0.1%) | 297.4 | 184 (21.7%) | |
| SMI_E12_NTC_rep01 | 60,385.00 (0.0%) | 284.8 | 23,356 | 47 (22.2%) |
| SMI_F12_NTC_rep02 | 64,471.00 (0.0%) | 304.1 | 24,663 | 47 (22.2%) |
| SMI_G12_NTC_rep03 | 69,440.00 (0.0%) | 327.5 | 26,829 | 42 (19.8%) |
| SMI_H12_NTC_rep04 | 57,911.00 (0.0%) | 273.2 | 22,004 | 48 (22.6%) |
| NC means | 63,051.75 (0.0%) | 297.4 | 24,213.0 | 24 (11.3%) |
SC samples are pooled plasma controls from an independent pooled plasma source that is different from IPCs. They could be a pooled sample derived from all study samples, for example. They should be from the same source across all plates that will be analysed together. SCs are used for calculating intra- and inter-plate coefficient of variation (CV). They are necessary for obtaining an unbiased measure of inter-plate CV when IPC normalization is used.
| Total count (%) | Mean count per target | Zeros n (%) | CV% mean | CV% median | |
|---|---|---|---|---|---|
| Total | 9,478,631 (3.0%) | 5,588.8 | 40 (2.4%) | 17.4% | 12.1% |
| SMI_A11_PP01_rep01 | 875,429 (0.3%) | 4,129.4 | 5 (2.4%) | ||
| SMI_B11_PP01_rep02 | 798,857 (0.2%) | 3,768.2 | 5 (2.4%) | ||
| SMI_C11_PP01_rep03 | 879,134 (0.3%) | 4,146.9 | 5 (2.4%) | ||
| SMI_D11_PP01_rep04 | 924,573 (0.3%) | 4,361.2 | 5 (2.4%) | ||
| SMI_E11_PP02_rep01 | 1,482,992 (0.5%) | 6,995.2 | 5 (2.4%) | ||
| SMI_F11_PP02_rep02 | 1,663,813 (0.5%) | 7,848.2 | 5 (2.4%) | ||
| SMI_G11_PP02_rep03 | 1,465,841 (0.5%) | 6,914.3 | 5 (2.4%) | ||
| SMI_H11_PP02_rep04 | 1,387,992 (0.4%) | 6,547.1 | 5 (2.4%) | ||
| Median | 1,156,082 (0.4%) | 5,453.2 | 5 (2.4%) |
Heatmaps show the percent relative to plate median for log2(total counts) and the specified internal control.
Plate-specific QC criteria:
QC criteria are assessed either on unnormalized (raw) or IC-normalized (IC) data.
| QC Criterion | Normalization | QC Status | QC Value | QC Threshold |
|---|---|---|---|---|
| ICRead_CV | raw | Fail | 55.3% | 50% |
| IPCRead_CV | raw | Pass | 10.5% | 50% |
| IPCTarget_CV | IC | Pass | 8.2% | 20% |
| Detectability | IC | Pass | 91.9% | 75% |
| MinReads | raw | Pass | 320,863,871 | 5e+05 |
Sample-specific QC criteria:
| # of Samples | Detectability | ICReads | NumReads | IC_Median | |
|---|---|---|---|---|---|
| Plate 1 | 80 | 1 | 0 | 0 | 1 |
| Total | 80 | 1 | 0 | 0 | 1 |
| Plate | QC_fail_samples |
|---|---|
| Plate 1 | SMI_G5_Donor65 |
| Plate | QC_fail_samples |
|---|---|
| Plate 1 | SMI_G5_Donor65 |
Target detectability is the percentage of samples that are above the limit of detection for that target. A target is considered “detectable” if it is above limit of detection in greater than 50% of samples. “Overall” detectability is the overall percentage of samples across all plates that are above LOD for a given target. Target detectability reported here excludes IPCs, SCs, NCs, and bridge samples.
| type | # samples | mean | sd | median | min | max | # of detectable targets (%) |
|---|---|---|---|---|---|---|---|
| all | 80 | 91.1 | 24.2 | 100 | 0 | 100 | 194 (91.9%) |
| all | |
|---|---|
| Activin AB | 65.0 |
| AGER | 100.0 |
| AGRP | 100.0 |
| ANGPT1 | 100.0 |
| ANXA1 | 76.2 |
| BDNF | 100.0 |
| BMP10 | 72.5 |
| BMP15 | 0.0 |
| BMP7 | 100.0 |
| C1QA | 100.0 |
| CCL1 | 100.0 |
| CCL13 | 100.0 |
| CCL14 | 100.0 |
| CCL15 | 100.0 |
| CCL16 | 100.0 |
| CCL17 | 100.0 |
| CCL19 | 100.0 |
| CCL2 | 100.0 |
| CCL20 | 100.0 |
| CCL22 | 100.0 |
| CCL23 | 100.0 |
| CCL24 | 100.0 |
| CCL25 | 100.0 |
| CCL28 | 100.0 |
| CCL3 | 22.5 |
| CCL4 | 100.0 |
| CCL5 | 100.0 |
| CCL7 | 98.8 |
| CCL8 | 100.0 |
| CD200 | 92.5 |
| CD200R1 | 100.0 |
| CD274 | 100.0 |
| CD276 | 100.0 |
| CD40 | 100.0 |
| CD70 | 83.8 |
| CD83 | 100.0 |
| CD93 | 100.0 |
| CEACAM5 | 100.0 |
| CHI3L1 | 100.0 |
| CLCF1 | 13.8 |
| CLEC4A | 100.0 |
| CSF1 | 100.0 |
| CSF2 | 0.0 |
| CSF2RB | 85.0 |
| CSF3 | 100.0 |
| CSF3R | 100.0 |
| CST7 | 100.0 |
| CTF1 | 100.0 |
| CTLA4 | 100.0 |
| CTSS | 100.0 |
| CX3CL1 | 100.0 |
| CXADR | 100.0 |
| CXCL1 | 100.0 |
| CXCL10 | 100.0 |
| CXCL12 | 100.0 |
| CXCL13 | 100.0 |
| CXCL14 | 98.8 |
| CXCL2 | 100.0 |
| CXCL3 | 100.0 |
| CXCL5 | 100.0 |
| CXCL8 | 100.0 |
| CXCL9 | 100.0 |
| EBI3 | 37.5 |
| EGF | 100.0 |
| EPO | 100.0 |
| FAM3C | 0.0 |
| FAM3D | 100.0 |
| FASLG | 100.0 |
| FGF19 | 100.0 |
| FGF2 | 93.8 |
| FGF21 | 100.0 |
| FGF23 | 100.0 |
| FGF5 | 0.0 |
| FLT1 | 100.0 |
| FLT3LG | 20.0 |
| FTH1 | 100.0 |
| GDF15 | 100.0 |
| GDF2 | 100.0 |
| GFAP | 100.0 |
| GRN | 100.0 |
| GZMB | 100.0 |
| HGF | 100.0 |
| IFNA1; IFNA13 | 98.8 |
| IFNA2 | 52.5 |
| IFNB1 | 95.0 |
| IFNG | 100.0 |
| IFNL1 | 100.0 |
| IFNW1 | 41.2 |
| IL10 | 100.0 |
| IL10RB | 100.0 |
| IL11 | 100.0 |
| IL12B | 100.0 |
| IL12p70 | 95.0 |
| IL12RB1 | 100.0 |
| IL13 | 100.0 |
| IL13RA2 | 100.0 |
| IL15RA | 100.0 |
| IL16 | 100.0 |
| IL17A | 98.8 |
| IL17B | 100.0 |
| IL17C | 100.0 |
| IL17F | 98.8 |
| IL17RB | 100.0 |
| IL18 | 100.0 |
| IL18BP | 100.0 |
| IL18R1 | 100.0 |
| IL19 | 98.8 |
| IL1B | 58.8 |
| IL1R1 | 0.0 |
| IL1R2 | 100.0 |
| IL1RL1 | 100.0 |
| IL1RN | 98.8 |
| IL20 | 100.0 |
| IL22 | 61.3 |
| IL23 | 98.8 |
| IL2RA | 100.0 |
| IL2RB | 100.0 |
| IL32 | 91.2 |
| IL33 | 98.8 |
| IL34 | 100.0 |
| IL35 | 2.5 |
| IL36A | 100.0 |
| IL36G | 91.2 |
| IL39 | 6.2 |
| IL3RA | 100.0 |
| IL4 | 100.0 |
| IL4R | 100.0 |
| IL5 | 100.0 |
| IL5RA | 100.0 |
| IL6 | 98.8 |
| IL6R | 100.0 |
| IL7 | 100.0 |
| IL7R | 100.0 |
| IL9 | 100.0 |
| INHBA | 87.5 |
| INHBB | 80.0 |
| Inhibin A | 25.0 |
| Inhibin B | 35.0 |
| IRAK4 | 100.0 |
| KDR | 100.0 |
| KITLG | 98.8 |
| LAG3 | 100.0 |
| LAMP3 | 98.8 |
| LCN2 | 100.0 |
| LGALS9 | 95.0 |
| LIF | 98.8 |
| LTBR | 100.0 |
| MDK | 100.0 |
| MERTK | 100.0 |
| MET | 100.0 |
| MICA | 85.0 |
| MICB | 100.0 |
| MIF | 100.0 |
| MMP10 | 100.0 |
| MMP12 | 98.8 |
| MMP3 | 100.0 |
| MMP8 | 100.0 |
| MMP9 | 100.0 |
| MPO | 100.0 |
| MUC16 | 100.0 |
| NCR1 | 100.0 |
| NGF | 100.0 |
| NIC_1 | 0.0 |
| NIC_3 | 0.0 |
| OSM | 100.0 |
| PDCD1 | 100.0 |
| PDGFA | 100.0 |
| PTX3 | 100.0 |
| S100A12 | 100.0 |
| S100A9 | 88.8 |
| SCG2 | 98.8 |
| SELE | 100.0 |
| SLURP1 | 100.0 |
| SPP1 | 100.0 |
| TAFA5 | 100.0 |
| TGFB1 | 100.0 |
| TGFB3 | 80.0 |
| THPO | 100.0 |
| TIMP1 | 100.0 |
| TIMP2 | 100.0 |
| TLR3 | 100.0 |
| TNF | 86.2 |
| TNFRSF11A | 100.0 |
| TNFRSF13C | 100.0 |
| TNFRSF14 | 100.0 |
| TNFRSF17 | 100.0 |
| TNFRSF18 | 100.0 |
| TNFRSF1A | 100.0 |
| TNFRSF1B | 100.0 |
| TNFRSF4 | 100.0 |
| TNFRSF8 | 100.0 |
| TNFRSF9 | 100.0 |
| TNFSF10 | 100.0 |
| TNFSF11 | 92.5 |
| TNFSF12 | 100.0 |
| TNFSF13 | 100.0 |
| TNFSF13B | 98.8 |
| TNFSF15 | 100.0 |
| TNFSF4 | 100.0 |
| TNFSF8 | 100.0 |
| TNFSF9 | 100.0 |
| TREM1 | 100.0 |
| TSLP | 100.0 |
| VCAM1 | 100.0 |
| VEGFA | 37.5 |
| VEGFC | 100.0 |
| VEGFD | 88.8 |
| VSNL1 | 100.0 |
| VSTM1 | 97.5 |
| WNT16 | 100.0 |
| WNT7A | 100.0 |
Plot shows target mCherry-IPC-normalized count distributions relative to LOD. For each target, log2(LOD) was subtracted from log2(count) for mCherry-normalized data.
%CV was calculated for each target (excluding internal controls) for the sample control replicates (if given) and IPCs. Only values above LOD were used to calculate %CV. If there are multiple plates, “Overall” represents the distribution for the target-specific mean intra-plate CV across all plates.
%CV was calculated for each target (excluding internal controls) for the sample control replicates (if given) and IPCs. Only values above LOD were used to calculate %CV.
| mean | sd | median | min | max | missing | |
|---|---|---|---|---|---|---|
| Plate 1 | 15.6 | 13.8 | 11.2 | 2.05 | 106 | 10 |
| mean | sd | median | min | max | missing | |
|---|---|---|---|---|---|---|
| Plate 1 | 12 | 3.67 | 11.7 | 1.48 | 30 | 14 |
| mean | sd | median | min | max | missing | |
|---|---|---|---|---|---|---|
| Plate 1 | 15.1 | 13.7 | 11.2 | 1.23 | 105 | 10 |
| mean | sd | median | min | max | missing | |
|---|---|---|---|---|---|---|
| Plate 1 | 8.77 | 3.81 | 8.08 | 1.89 | 26.8 | 14 |
Sample boxplots show distributions of the log2 counts for each sample, unnormalized and after mCherry + IPC normalization.
Plot shows the Pearson correlation between samples. Heirarchical clustering is done using complete linkage.
Hierarchical clustering of both samples and targets was done using a Euclidean distance metric and complete linkage. Data is log2 and mCherry-normalized; targets are centered and scaled.